This proposal is in response to RFA: PAS-10-151, a request for proposals to perform Alzheimer's disease drug discovery. Alzheimer's disease is a progressive and irreversible brain disorder with no known cure that affects over 30 million people worldwide with a large increase in incidence from 0.5% per year at the age of 65 to about 8% after the age of 85. In this disease, it is widely believed that the A[unreadable] protein, the major component of the observed extracellular senile plaques (amyloid fibrils) in the brains of Alzheimer's patients, is intimately and causally related to the disease. Recent evidence indicates that soluble oligomers of A[unreadable]42 may represent the primary toxic species of amyloid in AD. A class of small organic compounds has been discovered, namely tricyclic pyrones (TP), such as CP2 (code name), that prevents the death of human neuroblastoma MC65 cells related to intracellular accumulation of A[unreadable] oligomers, and nearly completely eliminated amyloid plaques in 5x familial Alzheimer's disease (5xFAD;a robust A[unreadable]42-producing animal model of AD) mice, after a eight-week course of oral administration of CP2 by oral gavage of 25 mg/kg (by weight), twice a day. This protective effect is intimately related to TP's ability to inhibit the formation of A[unreadable] aggregates, and we propose to study this effect by solid-state NMR structural measurements in order to develop structure-activity relationships and an understanding of how current TP candidates bind to and inhibit A[unreadable] oligomers. We will then use this information to design new, more effective tricyclic pyrone anti-Alzheimer's drug candidates that will be similarly evaluated spectroscopically and by neuronal cell protection assays. We propose the following specific aims: (1) identify the binding site of three TP candidates, with proven inhibitory effect, in the A[unreadable]42 oligomer:TP complex as well as investigate any differences in binding location and binding interactions by solid-state NMR;(2) study the inhibition of A[unreadable]42 aggregation as well as the disaggregation of A[unreadable] oligomers and protofibrils by each TP using additional spectroscopic techniques and atomic force microscopy;and (3) design and synthesize improved novel tricyclic pyrone drug candiates that will be evaluated by MC65 cell studies to confirm more effective inhibition of A[unreadable] aggregation and improved protection against cell death. PUBLIC HEALTH RELEVANCE: In Alzheimer's disease, it is widely believed that the A protein, the major component of the observed extracellular senile plaques (amyloid fibrils) in the brains of Alzheimer's patients, is intimately and causally related to the disease. A class of small organic compounds has been discovered, namely tricyclic pyrones, with exciting therapeutic characteristics including the apparent ability to disaggregate amyloid oligomers and early fibrils that has been demonstrated to significantly reduce amyloid plaques in mice with familial Alzheimer's disease. The goal of this project is to study how these compounds disaggregate amyloid and use the information learned to design and synthesize compounds that are able to block the formation of A[unreadable] lesions or reverse existing ones (an early step in the development of a drug for the treatment of Alzheimer's disease).